The present invention is directed to a process for separation of target cells from a sample.
Cell separation methods are widespread in scientific and clinical laboratories for research, diagnostics, or clinical applications. Most of these methods are limited to small amounts of cells only. For separation of cells from a vast number of non-target cells, magnetic cell sorting or gradient density centrifugation are established technologies.
A particular challenging separation procedure is the separation of cells from whole blood, for example T cells from whole blood. To this end, erythrocytes have to be discharged before isolating or purifying the target cells. Elimination of erythrocytes can be performed, for example, by gradient density centrifugation; peripheral blood mononuclear cells (PBMC) sample preparation, erythrocyte lysis, or aggregation by, for example, multifunctional antibodies, polysaccharides, polyvinylpyrrolidone, or polyoxyethylene and subsequent centrifugation. Discharging of erythrocytes prior to isolation of target cells is for example disclosed in EP2597153A1.
After the elimination of erythrocytes or further undesired cells, subsequent process steps for isolation the target cells need to be employed. Such methods are long known in the art and use, for example, monoclonal antibodies with affinity to cell surface antigens, for example as disclosed in U.S. Pat. No. 5,840,502, U.S. Pat. No. 5,648,223, U.S. Pat. No. 5,646,004, U.S. Pat. No. 5,474,687, or U.S. Pat. No. 7,316,932.
The use of multifunctional antibodies or polymers as aggregation agents is described, for example, in WO00/73794 or U.S. Pat. No. 7,160,723. The technique disclosed comprises contacting a sample containing nucleated cells and red blood cells with an aggregation agent, removal of the aggregated red blood cells by centrifugation and further purification of the nucleated cells using antibodies recognizing the desired cells, for example, by magnetic cell sorting. These methods have the disadvantage of several process steps including a time-consuming and laborious centrifugation step.
WO 2013/076070 teaches to deplete a plurality of non-target cells by aggregation and magnetic cell sorting to obtain a suspension of cells, which are not affected by the depletion cocktail. While the cells obtained with the process are “untouched”, the purity is rather low, because only a negative selection process is performed. It is not possible to obtain a specific, pure subpopulation of target cells with the process of WO 2013/076070. A similar process utilizing magnetic cells sorting is disclosed in WO01/17687A1 and WO96/31776A1.